P883: TARGETING CALCINEURIN/NFATC1 SIGNALING SENSITIZES PROTEASOME INHIBITION TREATMENT IN MULTIPLE MYELOMA
Authors: J. Zhang, X. Luo, T. Xia, K. C. Cheng, M. H. Ng
Journal: HemaSphere (2022)

Background: Proteasome inhibitors (PIs) (e.g., Bortezomib) are key treatments to improve the clinical outcome of multiple myeloma (MM). However, disease relapse from the emergence of PI-resistant clones is the main problem. Insights into the mechanisms underlying PI-resistance will provide novel druggable targets to prevent relapse. It was well-recognized that PIs induce endoplasmic reticulum (ER) stress in MM cells. ER stress is associated with the Calcineurin/nuclear-factor-of-activated-T-cells (NFAT) signaling. Accumulating studies indicated the importance of Calcineurin/NFAT signaling in tumorigenesis and drug resistance. However, the involvement of Calcineurin/NFAT signaling in PI-resistance in MM has not yet been investigated. Aims: To dissect the role of the Calcineurin/NFAT axis in Bortezomib (BTZ) treatment response and explore the therapeutic potential by combining Bortezomib and Calcineurin/NFAT inhibitors in MM. Methods: We studied the expression pattern of PPP3CA and NFATC1 from MM datasets and MM cell lines and their association with BTZ responses in MM. The expression of the four principal NFAT members was detected by expression microarray and their activation status by western blotting in MM cell lines. We identified the most prominent NFAT member and assessed its activation under BTZ treatment. A lentivirus-transduction system was used for overexpressing genes, and siRNA transfection was applied to induce NFATC1 knockdown in a BTZ-resistant cell line ARH77. Flow cytometry and western blotting assessed the combinational effect of Calcineurin/NFAT inhibitors and BTZ on MM cells. Results: The expression level of PPP3CA, which encodes Calcineurin Catalytic subunit A (CnA), was correlated to the poor clinical outcome of recurrent MM patients (MMRF cohort). Calcineurin was highly expressed in ARH77, which is less sensitive to BTZ treatment than other MM cell lines. Among the four NFAT members, we found that NFATC1 and NFATC3 were the most highly expressed members in MM cell lines. However, NFATc1 was the only member suppressed by CsA but activated by the calcium channel agonist Ionomycin in ARH77. Consistent with Calcineurin, NFATc1 was highly expressed in ARH77, with reference to a confirmed NFATc1-positive cell line Raji and the nuclear-NFATc1 overexpressing HEK-293T. Additionally, BTZ activated CnA and NFATc1 time-dependently in ARH77 but failed to activate CnA and NFATc1 in the BTZ-sensitive cell line NCI-H929. Immunofluorescence showed that BTZ triggered the nuclear translocation of NFATc1. From GEO datasets, NFATC1 was overexpressed in patients less responsive to BTZ, but was unchanged in patients subjected to conventional therapies. The Calcineurin/NFATc1 was thus a potential mechanism of BTZ-resistance in MM. Depleting NFATc1 enhanced BTZ-induced DNA damage-related apoptosis in ARH77. The combinational treatment of Calcineurin/NFAT inhibitors (CsA and FK506) and BTZ decreased the nuclear/cytosolic ratio of NFATc1 and promoted apoptosis in two MM cell lines compared to the treatment of BTZ alone. Image:Summary/Conclusion: The activation of the Calcineurin/NFATc1 axis is an important mechanism that contributes to BTZ-resistance in MM. Targeting this axis confers translational potential for improving BTZ-based therapies.